Long wavelength thiol-reactive fluorophores

ABSTRACT

Reactive fluorescent dyes compositions and methods of using same are disclosed. Nile Red nucleus dyes are disclosed having thiol-reactive groups. Nile Red nucleus dyes are disclosed that exhibit a fluorescence emission of at least about 575 nm. The Nile Red nucleus dyes are of the following formulae:

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Utility applicationSer. No. 11/131,283, filed May 18, 2005, which claims the benefit ofU.S. Provisional Application Ser. No. 60/573,944, filed May 21, 2004 andU.S. Provisional Application Ser. No. 60/599,514, filed Aug. 6, 2004,the contents of which are herein incorporated by reference in theirentirety.

This application is based on research work that was funded in part by agrant from the U.S. Army Medical Research and Material Command (USAMRMC)under TMM Contract No. W81XWH-04-1-0076 so that the United Statesgovernment may have certain rights in this invention.

FIELD OF THE INVENTION

The embodiments of the present invention are directed to novel longwavelength fluorophores for use in the detection of an analyte.Additional embodiments are directed to fluorophores that contain athiol-reactive site that can be covalently attached to a thiol group ofa molecule.

SUMMARY OF THE EMBODIMENTS

Fluorescent dyes or fluorophore compounds are suitable for use invarious chemical and biological processes. Various embodiments aredirected to fluorophores having a reactive group that can be used tocouple or conjugate the fluorophore with another molecule such as aprotein. Biosensors comprising fluorophores having a reactive groupcoupled or conjugated with a protein.

Additional embodiments are fluorophores having a reactive group and thathave an emission wavelength of not less than about 575 nm, referred toas near-infrared dyes (NIR dyes). In one embodiment, the fluorophoreshave an emission at about 650 nm. The fluorophore embodiments include apendant reactive group capable of conjugating with a member of aspecific binding pair.

The fluorophores are suitable for coupling to receptors and to bindingproteins having an affinity for a specific ligand or analyte. In variousembodiments of the invention, the receptor or binding protein undergoesconformational changes when coupled to the ligand or analyte. Thefluorophores when coupled to the binding protein exhibit a detectablesignal change as a result of binding of ligand.

Another embodiment provides a fluorophore having a reactive moiety thatcan be covalently attached to an amino acid. The fluorophores in oneembodiment have a thiol-reactive group that can be conjugated to acysteine residue of a protein amino acid. Examples of suitablethiol-reactive groups that can be introduced into the fluorophoreinclude a halo-acetyl and particularly an iodoacetyl group. Otherthiol-reactive groups include iodoacetamide, bromoacetamide, iodoacetateor maleimide.

A further embodiment of the invention provides a fluorophore having athiol-reactive group and having an emission of at least about 575 nm.The fluorophores in one embodiment of the invention are benzodioxazole,squaraine, 9-diethylamino-5H-benzo phenoxazin-5-one (hereinafterreferred to as Nile Red), coumarin, and aza coumarin. In anotherembodiment, the invention is directed to derivatives of squaraine,benzodioxazole, Nile Red, coumarin and aza coumarin, hereinafterreferred to interchangeably as squaraine nucleus or nuclei,benzodioxazole nucleus or nuclei, Nile Red nucleus or nuclei, coumarinnucleus or nuclei and aza coumarin nucleus or nuclei, respectively, orcollectively as “fluorescent dye.” Derivatives of the squaraine nuclei,benzodioxazole nuclei, Nile Red nuclei, coumarin nuclei and aza coumarinnuclei include any reaction product of the derivative, for example, witha protein amino acid group. Derivative is meant to include any chemicalmodification, addition, deletion, or substitution to an aforementionednucleus. One embodiment includes nuclei of the aforementioned dyes thatexhibit a fluorescence emission of at least about 575 nm are included asembodiments. In one embodiment, the squaraine nuclei, benzodioxazolenuclei, Nile Red nuclei, coumarin nuclei and aza coumarin nuclei containa thiol-reactive group for binding to a protein.

Another embodiment is also directed to a conjugate of a binding proteinand a squaraine nucleus, benzodioxazole nucleus, Nile Red nucleus,coumarin nucleus and aza coumarin nucleus coupled to the binding proteinthrough a cysteine residue on the binding protein. The cysteine residueof the protein can be naturally occurring or engineered into theprotein. In one embodiment, the binding protein is a glucose bindingprotein that has an affinity for glucose and reversibly binds glucose.The fluorophore produces a detectable change in a fluorescence propertyin response to binding. The detectable change in a fluorescent propertycan be a shift in the wavelength of emission, a change in intensity ofthe emitted energy, a change in fluorescence lifetime, a change inanisotropy, change in polarization, or a combination thereof. In anotherembodiment, the binding protein is a maltose binding protein (MBP) thathas an affinity for and binds maltose. In another embodiment, thebinding protein is altered so that it has an affinity for and bindsnon-native ligands.

The various embodiments of the present invention provide for afluorophore having the formulaA-Ywhere A is selected from the group consisting of squaraine nucleus, NileRed nucleus, benzodioxazole nucleus, coumarin nucleus, and an azacoumarin nucleus, and where Y is

where n is an integer of 1 to 6, or Y is A′-CO—R¹, where A′ is —R²O— or—R²N(R³)—, where R² is a C₁ to C₆ alkyl, R³ is H or CH₃, and R¹ isCH₂Cl, CH₂Br, CH₂I, or

where m is an integer of 2 to 6.

Additional embodiments provide for a biosensor compound having theformulaA-Y′—Bwhere A is a fluorophore selected from the group consisting of asquaraine nucleus, a Nile Red nucleus, a benzodioxazole nucleus, acoumarin nucleus, and an aza coumarin nucleus, where Y′—B is

where n is an integer of 1 to 6, or Y′—B is A′-CO—V—B, where A′ is —R²O—or —R²N(R³)— where R² is a C₁ to C₆ alkyl, R³ is H or CH₃, and V—B is—CH₂—B or

where m is an integer of 2 to 6, and B is a receptor having a reversiblebinding affinity for a ligand to be detected, and where the biosensorcompound exhibits a detectable change in a fluorescence property as aresult of changes in concentration of the ligand.

Further embodiments are methods for detecting analyte comprising:providing a biosensor compound having at least one mutated bindingprotein with a fluorophore covalently bonded thereto through a thiolgroup of said binding protein, where the fluorophore exhibits anemission fluorescence of at least 575 nm and is selected from the groupconsisting of a squaraine nucleus, Nile Red nucleus, benzodioxazolenucleus, and aza coumarin nucleus. The biosensor compound is subjectedto an energy source to excite said fluorophore and to detect afluorescence property as an indicator of a analyte concentration in theanalyte-containing source.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings, in which:

FIG. 1 is a graph showing the absorbance spectrum of compound 2 inchloroform;

FIG. 2 is a graph showing the excitation and emission spectra ofcompound 2 in chloroform;

FIG. 3 is a titration curve of compound 1 conjugated to H152C GGBP inPBS buffer;

FIG. 4 is a graph showing the absorbance spectrum of compound 2conjugated to V19C GGBP in PBS buffer;

FIG. 5 is a graph showing the change in fluorescence of compound 2conjugated to V19C GGBP in response to a change in glucose concentrationin PBS buffer;

FIG. 6 is a schematic view of instrumentation used in Example 15; and

FIG. 7 illustrates a change in a fluorescence property of 9 conjugatedto A213C GGBP upon addition of glucose in an in vitro through skinmeasurement.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present disclosure is directed to fluorescent dyes that are suitablefor use as components of biosensors for detecting a ligand andparticularly an analyte, and methods of use. One embodiment is directedto fluorescent dyes that can be conjugated to a receptor to detect,quantify, or detect and quantify the ligand.

In one embodiment, the fluorescent dyes are used to produce a biosensorwhere the fluorescent dye is covalently attached to a binding protein.As used herein, the term “biosensor” and “biosensor compound” refers toa compound that undergoes a detectable change in specific response to aligand or target analyte. The embodiments of the biosensor discussedherein include a binding protein that is capable of binding to ananalyte. In other embodiments, the biosensor of the invention is able todetect an analyte and to detect changes in the analyte concentration. Invarious embodiments, the protein may be chosen from the group ofperiplasmic binding proteins that includes, but is not limited to,glucose/galactose binding protein, maltose binding protein,allose-binding protein, arabinose-binding protein, dipeptide-bindingprotein, glutamic acid/aspartic acid-binding protein, glutamine-bindingprotein, Fe(III)-binding protein, histidine-binding protein,leucine-binding protein, leucine/isoleucine/valine-binding protein,lysine/arginine/ornithine-binding protein, molybdate-binding protein,oligopeptide-binding protein, phosphate-binding protein, ribose-bindingprotein, sulfate-binding protein, Zn(II)-binding protein, and vitaminB-12-binding protein.

In other embodiments, the biosensor is a fluorescent dye covalentlyattached to a binding protein, wherein the protein-dye conjugateexhibits a fluorescence emission of 575 nm or higher. In one embodiment,the fluorescent dye exhibits a fluorescence emission of not less than575 nm. In one exemplary form, the binding protein is aglucose/galactose binding protein (GGBP) that is able to bind withglucose when in contact with a glucose-containing source. In anotherembodiment, the binding protein is a maltose binding protein (MBP). Notto be held by any theory, the binding protein is understood to undergo aconformational change upon binding of ligand. The percentage of bindingprotein binding sites occupied by ligand is dependent upon theconcentration of ligand and the binding constant of the binding protein.

The fluorescent dye embodiments that exhibit a fluorescence emission ofat least about 575 nm avoid or minimize background interference from thebiological system or other components in the glucose source. Thefluorescent dyes exhibit a change in intensity of the fluorescencesignal, a shift in the emission wavelength of the maximum fluorescenceemission, a change in fluorescence lifetime, a change in anisotropy, achange in polarization, or a combination thereof, when the bindingprotein undergoes a conformational change as a result of changes in theglucose concentration. In the method embodiment, the biosensor contactsa sample containing analyte, for example glucose, to enable the analyteto bind with the binding protein, where the sample includes, but is notlimited to, blood, saliva, interstitial fluid, etc. An energy source,such as a laser or LED, is applied to the biosensor to excite thefluorescent dye, and a fluorescence property is detected. Due to eithera conformational change in the binding protein, subsequent changes inthe microenvironment of the dye, or both, the detected fluorescenceproperty or change of the detected fluorescence property can becorrelated to the presence of an analyte or a analyte concentration. Thefluorescence and detection can be carried out continuously orintermittently at predetermined times. Thus, episodic or continuoussensing of analyte, for example, glucose, is envisaged. The biosensordisclosed herein is adaptable for use in strips, implants, micro- andnano-particles, and the like.

The fluorescent dye is covalently attached to the binding protein in asite-specific manner to obtain the desired change in the fluorescence.The fluorescent dye is attached at a site on the binding protein so thatthe conformational change maximizes the change in fluorescenceproperties. In other embodiments of the invention, the fluorescent dyeshave a thiol-reactive group that can be coupled to the thiol group on acysteine residue of the binding protein. The fluorescent dye includesthe aforementioned derivatives of the squaraine nuclei, benzodioxazolenuclei, Nile Red nuclei, coumarin nuclei and aza coumarin nuclei.

The biosensor in one embodiment has the formula IA-Y′—B   (I)

In the formula I, A is squaraine nucleus, Nile Red nucleus,benzodioxazole nucleus, coumarin nucleus, aza coumarin nucleus, andderivatives thereof, Y′—B is

where n is an integer of 1 to 6, or Y′—B is A′-CO—V—B, where A′ is —R²O—or —R²N(R³)—. R² is a C₁ to C₆ alkyl. In one embodiment, R² is a C₂ toC₄ alkyl. R³ is H or CH₃. V—B is —CH₂—B or

where m is an integer of 2 to 6. In one embodiment, R²O is —CH₂CH₂O—. Inanother embodiment, R²N(R³) is —CH₂CH₂NH—. B is a receptor having abinding affinity for a ligand being detected and monitored by thebiosensor. The biosensor compound exhibits a detectable change in afluorescence property as a result of changes in concentration of theligand. In one embodiment, B is a glucose/galactose binding protein thatexhibits a detectable change in fluorescence emission as a result ofchanges in concentration of the ligand such as glucose. In anotherembodiment, B is a maltose binding protein.

In one embodiment, fluorescent dyes or fluorophores have athiol-reactive group and have the general formulaA-Y   (II)where A is a squaraine nucleus, Nile Red nucleus, benzodioxazole nucleusnucleus, coumarin nucleus, aza coumarin nucleus, or derivative thereofand Y is a thiol-reactive group.

In other embodiments, in formula II, Y is

where n is an integer of 1 to 6, or Y is A′-CO—R¹. A′ is —R²O— or—R²N(R3)—. R² is a C₁ to C₆ alkyl. In one embodiment, R² is a C₂ to C₄alkyl. R³ is H or CH₃. R¹ is CH₂Cl, CH₂Br, —CH₂I, or

where m is an integer of 2 to 6. The dye may exhibit a fluorescenceemission of at least about 575 nm. In one embodiment, R²O is —CH₂CH₂O—.In another embodiment, R²N(R³) is —CH₂CH₂NH—. In a further embodiment, Yis —(CH₂)₂OCOCH₂CH₂X, where X is Cl, Br or I. In other embodiments, R²is a C₂ to C₄ alkyl such as —CH₂CH₂—.

The squaraine nucleus as embodiments of the invention are based onderivatives of the squaraine structure

The squaraine nucleus of formula III exhibits changes in itsfluorescence properties with changes in its environment. For example,the squaraine nucleus III exhibits a 20-fold increase in itsfluorescence quantum yield by changing its environment from a polarprotic solvent such as methanol to a non-polar solvent such as toluene[C. Cornelissen-Gude, W. Rettig, R. Lapouyade. “Photophysical propertiesof Squaraine Derivatives: Evidence for Charge Separation.” J. Phys.Chem. A 1997, 101, 9673-9677]. Squaraine dyes can have an absorbancemaximum near 635 nm and exhibit a fluorescence emission peak at about650 nm. These dyes can fluoresce readily when exposed to the light froma red laser diode excitation source, for example.

The thiol-reactive squaraine nucleus embodiments of the invention havethe structures of formula IV and formula V

where R′ is H or OH, R″ are independently methyl, ethyl or propyl, R⁴ isa C₁ to C₆ alkyl or (CH₂)_(q)CO₂H, where q is an integer of 1 to 5, Zand Z′ are independently S, O, or C(CH₃)₂, W and W′ are independently H,CH₃, SO₃H, fused benzene, or fused sulfobenzene, and Y is as previouslydefined.

In one embodiment, the thiol-reactive squaraine nucleus has the formula

where R⁴ is ethyl in compound 2 or (CH₂)₂CO₂H in compound 3.

Another embodiment includes Nile Red nuclei generally having anabsorbance of about 550 nm and emission maxima of about 575 nm or more.These nuclei typically exhibit a shift of the emission maxima to as muchas 650 nm in lipid environments. In one embodiment, Nile Red nuclei havethe formula

where R⁵, R⁶, and R⁷ are independently methyl, ethyl or propyl, and Y isas previously defined.

In one embodiment, the Nile Red nucleus has the formula

One embodiment includes benzodioxazole nuclei having the formula

where r is an integer of 1 to 3, R⁸ and R⁹ are independently a C₁ to C₆alkyl or (CH₂)_(s)CO₂H, where s is an integer of 2 to 5. Z is S, O, orC(CH₃)₂. W is H, CH₃, SO₃H, fused benzene, or fused sulfobenzene. Y isas previously defined.

In one embodiment, the benzodioxazole nucleus has the formula

Another embodiment includes coumarin and aza coumarin nuclei having theformula

where D is CH or N, r is an integer of 1 to 3, R¹⁰, R¹¹ and R¹² areindependently C₁ to C₆ alkyl or (CH₂)_(s)CO₂H, where s is an integer of2 to 5, Z is S, O or C(CH₃)₂. W is H, CH₃, SO₃H, fused benzene or fusedsulfobenzene and Y is as previously defined.

In one embodiment, the aza coumarin nucleus has the formula

The squaraine nucleus embodiments of the invention can be synthesized byvarious known techniques. Symmetrical nuclei can be prepared by reactingan aromatic nucleophile with squaric acid. A first reaction scheme forproducing an iodoacetyl squaraine is depicted in the Scheme I asfollows.

A second reaction scheme for producing an iodoacetamidyl squarainenucleus derivative is depicted in the Scheme II as follows.

The Nile Red nucleus derivative embodiments can also be prepared usingvarious reaction schemes. A first reaction scheme for producing aniodoacetyl derivative is depicted in the Scheme III as follows.

A second reaction scheme for producing an iodoacetamidyl derivative isdepicted in the Scheme IV as follows.

A procedure for producing the benzodioxazole nucleus derivativeembodiments is depicted in the reaction Scheme V as follows.

Scheme V can be modified to produce benzodioxazole nucleus derivativescontaining other ring systems, as shown below in Scheme VI.

An alternative procedure for producing benzodioxazole nucleusderivatives is shown in reaction scheme VIa.

An exemplary procedure for the synthesis of benzodioxazole nucleus withan iodoacetyl linker is depicted in Scheme VII

An alternative procedure for producing benzodioxazole nucleusderivatives is shown in the reaction Scheme VIIa.

An exemplary procedure for producing the aza coumarin nucleusderivatives is depicted in the reaction Scheme VIII.

An alternative procedure for producing the aza coumarin nucleusderivatives is depicted in reaction Scheme VIIIa.

Another exemplary procedure for producing coumarin nuclei is depicted inreaction Scheme IX.

Another procedure for producing the coumarin nucleus derivatives isdepicted in reaction Scheme IXa.

In another embodiment of the invention, benzodioxazole, squaraine, NileRed, coumarin, and aza coumarin nuclei have a fluorescence emission. Inone embodiment, the specific fluorescent nuclei described above havefluorescent emission above about 575 nm.

In one embodiment, the resulting thiol-reactive nuclei are reacted witha binding protein to produce fluorescent binding protein conjugatesuseful as biosensors.

In one embodiment the resulting squaraine nucleus-binding proteinconjugates have the Markush formulas:

where Y′—B is

where n is an integer of 1 to 6, or Y′—B is A′-CO—V—B, where A′ is —R²O—or —R²N(R³)—. R² is a C₁ to C₆ alkyl. In one embodiment, R² is a C₂ toC₄ alkyl. R³ is H or CH₃. V—B is —CH₂—B or

where m is an integer of 2 to 6. In one embodiment, R²O is —CH₂CH₂O—. Inanother embodiment, R²N(R³) is —CH₂CH₂NH—, and where B is bindingprotein.

In one embodiment the resulting Nile Red nucleus-binding proteinconjugate has the Markush formulas:

where Y′B is as previously defined and B is binding protein.

In one embodiment the resulting benzodioxazole nucleus-binding proteinconjugate has the Markush formulas:

where Y′B is as previously defined and B is binding protein.

In one embodiment the resulting coumarin, and aza coumarinnucleus-binding protein conjugate has the Markush formulas:

where Y′B is as previously defined and B is binding protein.

In one embodiment the resulting binding protein conjugate has theformula:

The binding proteins B include a thiol group, for example, a cysteineresidue, that is able to react with the thiol-reactive fluorescent dye.The term “binding proteins” refers to proteins that interact withspecific analytes in a manner capable of transducing or providing adetectable signal differentiable either from when analyte is notpresent, analyte is present in varying concentrations over time, or in aconcentration-dependent manner, by means of the methods described.Capable of transducing or “provide a detectable signal”, as used herein,refers to the ability to recognize a change in a property of a reportergroup in a manner that enables the detection of ligand-protein binding.For example, in one embodiment, the mutated GGBPs comprise a detectablereporter group whose detectable characteristics alter upon glucosebinding. The change in the detectable characteristics may be due to analteration in the environment of the label attached to the mutated GGBPor a conformational change of the protein resulting from binding. Thetransducing or providing a detectable signal may be reversible ornon-reversable. As used herein, the terms transducing and providing adetectable signal are used interchangeably. The transduction eventincludes continuous, programmed, and episodic means, including one-timeor reusable applications. Reversible signal transduction may beinstantaneous or may be time-dependent, providing a correlation with thepresence or concentration of analyte is established. Binding proteinsmutated in such a manner to effect transduction are embodiments of thepresent invention. Binding proteins include, but are not limited to,glucose/galactose-binding protein, maltose-binding protein,allose-binding protein, arabinose-binding protein, dipeptide-bindingprotein, glutamic acid/aspartic acid-binding protein, glutamine-bindingprotein, Fe(III)-binding protein, histidine-binding protein,leucine-binding protein, leucine/isoleucine/valine-binding protein,lysine/arginine/ornithine-binding protein, molybdate-binding protein,oligopeptide-binding protein, phosphate-binding protein, ribose-bindingprotein, sulfate-binding protein, Zn(II)-binding protein, and vitaminB-12-binding protein.

The term “glucose/galactose binding protein” or “GGBP” or “maltosebinding protein” or “MBP” as used herein refers to a type of proteinnaturally found in the periplasmic compartment of bacteria. Theseperiplasmic proteins are naturally involved in chemotaxis and transportof small molecules (e.g., sugars, amino acids, and small peptides) intothe cytoplasm. For example, GGBP is a single chain protein consisting oftwo globular α/β domains that are connected by three strands to form ahinge. The binding site is located in the cleft between the two domains.When glucose enters the binding site, GGBP undergoes a conformationalchange, centered at the hinge, which brings the two domains together andentraps glucose in the binding site. The wild type E. coli GGBP DNA andamino acid sequence can be found atwww.ncbi.nlm.nih.gov/entrez/accession number D90885 (genomic clone) andaccession number 23052 (amino acid sequence). In one embodiment, GGBP isfrom E. coli.

“Mutated binding protein” (for example “mutated GGBP”) as used hereinrefers to binding proteins from bacteria wherein at least one amino acidhas been substituted for, deleted from, or added to, the protein.

Mutations of binding proteins include for example, the addition orsubstitution of cysteine groups, non-naturally occurring amino acids,and replacement of substantially non-reactive amino acids with reactiveamino acids.

Additional embodiments are mutations of the GGBP protein having acysteine substituted for lysine at position 11 (K11C), a cysteinesubstituted for aspartic acid at position 14 (D14C), a cysteinesubstituted for valine at position 19 (V19C), a cysteine substituted forasparagine at position 43 (N43C), a cysteine substituted for glycine atposition 74 (G74C), a cysteine substituted for tyrosine at position 107(Y107C), a cysteine substituted for threonine at position 110 (T110C), acysteine substituted for serine at position 112 (S 112C), a doublemutant including a cysteine substituted for serine at position 112 andserine substituted for leucine at position 238 (S112C/L238S), a cysteinesubstituted for lysine at position 113 (K113C), a cysteine substitutedfor lysine at position 137 (K137C), a cysteine substituted for glutamicacid at position 149 (E149C), a double mutant including a cysteinesubstituted for glutamic acid at position 149 and an argininesubstituted for alanine at position 213 (E149C/A213R), a double mutantincluding a cysteine substituted for glutamic acid at position 149 and aserine substituted for leucine at position 238 (E149C/L238S), a doublemutant including a serine substituted for alanine at position 213 and acysteine substituted for histidine at position 152 (H152C/A213S), acysteine substituted for methionine at position 182 (M182C), a cysteinesubstituted for alanine at position 213 (A213C), a double mutantincluding a cysteine substituted for alanine at position 213 and acysteine substituted for leucine at position 238 (A213C/L238C), acysteine substituted for methionine at position 216 (M216C), a cysteinesubstituted for aspartic acid at position 236 (D236C), a cysteinesubstituted for leucine at position 238 (L238C) a cysteine substitutedfor aspartic acid at position 287 (D287C), a cysteine substituted forarginine at position 292 (R292C), a cysteine substituted for valine atposition 296 (V296C), a triple mutant including a cysteine substitutedfor glutamic acid at position 149 and a serine substituted for alanineat position 213 and a serine substituted for leucine at position 238(E149C/A213S/L238S), a triple mutant including a cysteine substitutedfor glutamic acid at position 149 and an arginine substituted foralanine at position 213 and a serine substituted for leucine at position238 (E149C/A213R/L238S), a cysteine substituted for glutamic acid atposition 149 and a cysteine substituted for alanine at position 213 anda serine substituted for leucine at position 238 (E149C/A213C/L238S).Additional embodiments include mutations of GGBP at Y10C, N15C, Q26C,E93C, H152C, M182C, W183C, L255C, D257C, P294C, and V296C.

Additional embodiments are mutations of the maltose binding proteinincluding, for example, D95C, F92C, I329C, S233C, and S337C.

Additional embodiments are histidine binding protein including, forexample, E167C, K229C, V163C, Y230C, F231C, and Y88C.

Additional embodiments are mutations of the sulfate-binding proteinincluding, for example, L65C, N70C, Q294C, R134C, W290C, and Y67C.

Additional embodiments are arabinose-binding protein including, forexample, D275C, F23C, K301C, L253C, and L298C.

Additional embodiments are mutations of the dipeptide-binding proteinincluding, for example, D450C, K394C, R141C, S111C, T44C, and W315C.

Additional embodiments are mutations of the glutamic acid/asparticacid-binding protein including, for example, A207C, A210C, E119C, F126C,F131C, F270C, G211C, K268C, Q123C, and T129C.

Additional embodiments are mutations of the glutamine-binding proteinincluding, for example, N160C, F221C, K219C, L162C, W220C, Y163C, andY86C.

Additional embodiments are mutations of the Fe(III)-binding proteinincluding, for example, E203C, K202C, K85C, and V287C.

Additional embodiments are mutations of the ribose-binding proteinincluding, for example, T135C, D165C, E192C, A234C, L236C, and L265C.

Additional embodiments are mutations of the phosphate-binding proteinincluding, for example, A225C, N223C, N226C, S164C, S39C, and A197C.

The mutation may serve one or more of several purposes. For example, anaturally occurring protein may be mutated in order to change thelong-term stability of the protein, to conjugate the protein to aparticular encapsulation matrix or polymer, to provide binding sites fordetectable reporter groups, to adjust its binding constant with respectto a particular analyte, or combinations thereof. Long-term stability isintended to include thermal stability.

In one embodiment, analyte and mutated protein act as binding partners.The term “associates” or “binds” as used herein refers to bindingpartners having a relative binding constant (Kd) sufficiently strong toallow detection of binding to the protein by a detection means. The Kdmay be calculated as the concentration of free analyte at which half theprotein is bound, or vice versa. When the analyte of interest isglucose, the Kd values for the binding partners are between about 0.0001mM and about 50 mM.

The fluorescent label can be attached to the mutated protein, forexample a GGBP, by any conventional means known in the art. For example,the reporter group may be attached via amines or carboxyl residues onthe protein. Exemplary embodiments include covalent coupling via thiolgroups on cysteine residues of the mutated or native protein. Forexample, for mutated GGBP, cysteines may be located at position 10, atposition 11, position 14, at position 15, position 19, at position 26,at position 43, at position 74, at position 92, at position 93, position107, position 110, position 112, at position 113, at position 137, atposition 149, at position 152, at position 154, at position 182, atposition 183, at position 186, at position 211, at position 213, atposition 216, at position 238, at position 240, at position 242, atposition 255, at position 257, at position 287, at position 292, atposition 294, and at position 296.

Any thiol-reactive group known in the art may be used for attachingreporter groups such as fluorophores to the cysteine in a natural or anengineered or mutated protein. For example, iodoacetamide,bromoacetamide, or maleimide are well known thiol-reactive moieties thatmay be used for this purpose.

Fluorophores that operate at long emission wavelengths (for example,about 575 nm or greater) are embodiments when the molecular sensor is tobe used in vivo, for example, incorporated into an implantable biosensordevice (the skin being opaque below about 575 nm). Conjugates containingthese fluorophores, for example, attached at various cysteine mutantsconstructed in mutated GGBPs, can be screened to identify which onesresult in the largest change in fluorescence upon glucose binding.

The following examples demonstrate the various embodiments of theinvention.

Example 1

In this example, the process of Scheme I was used to produce iodoestersquaraine nucleus 1.

Intermediate 1c: A mixture of butanol (5 mL) and benzene (5 mL) wasadded to a flask containing N-(2-hydroxyethyl)-N-methylaniline 1a (332mg, 2.2 mmol) and4-(N,N-dimethylaminophenyl)-3-hydroxy-cyclobuten-1,2-dione 1b (434 mg,2.0 mmol). The mixture was heated to reflux, and 5 g of 4 Å molecularsieves were added. After 1 h the mixture turned a light blue green.After 24 h the mixture was a dark green-blue suspension. The solvent wasevaporated, and the residue was washed with ethyl acetate (25 mL). Theremaining residue was washed with water, leaving a dark blue residue.This mixture was transferred to a soxhlet thimble and extractedovernight with methylene chloride. After evaporation the residue waswashed with ethyl acetate and yieldedN-(2-hydroxyethyl)-N,N′,N′-trimethyl-bis(4-aminophenyl)-squaraine 1c asa dark blue solid (223 mg, 30%).

Compound 1: One drop of DMF was added to a solution of iodoacetic acid(224 mg, 1.2 mmol) in methylene chloride (5 mL), and the resultingsolution was added dropwise over a 1 min period to oxalyl chloride (180mg, 1.4 mmol). After vigorous bubbling subsided, the pale orangesolution was stirred for 30 min at 25° C. The solvent was removed undervacuum (˜14 mm Hg), and the dark orange residue was kept under vacuumfor 15 min. This residue was dissolved in methylene chloride (5 mL) toprovide a solution of iodoacetyl chloride. The hydroxyethyl squaraine 1cfrom the preceding synthesis (175 mg, 0.5 mmol) was suspended inmethylene chloride (20 mL). N,N-diisopropylethylamine (154 mg, 1.2 mmol)and the previously prepared iodoacetyl chloride solution were added tothe squaraine suspension in sequential portions. The resulting blueheterogeneous mixture was stirred at 25° C. for 2 h. The solvent wasevaporated, and the resulting dark blue solid was washed six times with10 mL of ethylacetate, six times with ethyl ether, and dried undervacuum. This provided 204 mg of the iodoacetyl ester 1 as a palepurplish-blue solid. Fluorescence spectrum (methanol): 643 nm excitationmaximum, 669 nm emission maximum.

Example 2

In this example, iodoacetamide squaraine nucleus 2 was produced by theprocess in Scheme II. This example corresponds to Scheme II where R isC₂H₅.

Intermediate 2b: 2-methylbenzothiazole (1 mM) 2a andN-bromoethylphthalimide (1 mM) were heated in a round bottom flask at100° C. for 24 h. The resultant solids were isolated by filtration andpurified by continuous washing with chloroform to obtain theintermediate 2b.

¹H NMR (CD₃OD, TMS) δ ppm: 4.295 (t, CH₂, 2H); 4.871 (s, CH₃, 3H); 5.102(t, CH₂, 2H); 7.733 and 8.148-8.279 (m, aromatic, 8H).

Mol. Wt calculated for C₁₈H₁₅N₂O₂S is 323(M+), found 323 (FAB)

Intermediate 2c: Dibutyl squarate was reacted with3-ethyl-2-methyl-benzothiazolium iodide (1:1 ratio) in ethanol atreflux. After 30 minutes refluxing, the reaction mixture was filteredwhile hot. An orange colored solid crystallized out of the filtratewhile cooling, which was separated and re-suspended in ethanol andtreated with 40% NaOH solution under reflux. After 30 minutes, thecontents were cooled and acidified with 2 N HCl (pH adjusted to 4). Theproduct was extracted with chloroform to give intermediate 2c.

¹H NMR (CDCl₃, TMS) δ ppm: 1.385 (t, CH₃, 3H); 1.456 (t, CH₃, 3H); 4.064(q, CH₂, 2H); 4.794 (q, CH₂, 2H); 5.479 (s, CH, 1H); 7.026-7.518 (m,aromatic, 4H).

Mol. Wt calculated for C₁₄H₁₁NO₃S is 273(M+), found 273 (FAB)

Intermediate 2d: Intermediate 2b and 2c were reacted in a solventmixture containing 1:1 (v/v) toluene and n-butanol. The reaction mixturewas subjected to azeotropic distillation, and the water formed duringthe reaction was removed using a Dean-Stark trap. After 6 h, thereaction mixture was allowed to cool to room temperature, and the blueproduct was filtered out. Further purification was carried out usingflash column chromatography over silica gel using methanol andchloroform (1:4 ratio) as eluent to obtain dye 2d.

¹H NMR (CDCl₃, TMS) δ ppm: 1.439 (t, CH₃, 3H); 4.127 (t, CH₂, 2H); 4.181(q, CH₂, 2H); 4.381 (t, CH₂, 2H); 5.924 (s, CH, 1H); 5.964 (s, CH, 1H);7.070-7.823 (m, aromatic, 12H).

Mol. Wt calculated for C₃₂H₂₃N₃O₄S₂ is 577(M+), found 577 (FAB).

Intermediate 2e: The dye 2d was dissolved in anhydrous methylenechloride and was treated with hydrazine monohydrate at room temperature.The deprotected squaraine dye precipitated from the reaction mixture andwas isolated by filtration, followed by repeated washing with methylenechloride.

¹H NMR (CD₃OD, TMS) δ ppm: 1.420 (t, CH₃, 3H); 3.780 (t, CH₂, 2H); 4.320(q, CH₂, 2H); 4.520 (t, CH₂, 2H); 5.924 (s, CH, 1H); 5.964 (s, CH, 1H);7.200-7.850 (m, aromatic, 8H).

Mol. Wt calculated for C₂₄H₂₁N₃O₂S₂ is 447(M+), found 447 (FAB)

Compound 2: The intermediate dye 2e was dissolved in anhydrous methylenechloride, and an equivalent amount of iodoacetic anhydride was added.The mixture was stirred for 3 h at room temperature, and the product wasisolated by evaporation of the solvent to obtain the final dye 2.Purification was carried out by repeated precipitation fromhexane/methylene chloride.

¹H NMR (CD₃OD, TMS) δ ppm: 1.420 (t, CH₃, 3H); 3.900 (s, CH₂, 2H); 4.300(q, CH₂, 2H); 4.450 (t, CH₂, 2H); 4.480 (t, CH₂, 2H); 5.820 (s, CH, 1H);5.980 (s, CH, 1H); 7.200-8.200 (m, aromatic, 8H).

Mol. Wt calculated for C₂₆H₂₂IN₃O₃S₂ is 615(M+), found 615 (FAB).

An illustrative absorbance spectrum is shown in FIG. 1 for compound 2 inchloroform. The excitation and emission spectra of compound 2 inchloroform are shown in FIG. 2.

Example 3

In this example, the process of Example 2 was repeated except for Rbeing CH₂CH₂CO₂H of intermediate 3c in Scheme II. The resultingintermediate dye 3d with the protected amino group was characterized byNMR spectroscopy.

¹H NMR (DMSO-d₆, TMS) δ ppm: 2.720 (t, CH₂, 2H); 3.970 (t, CH₂, 2H);4.470 (t, CH₂, 2H); 4.530 (t, CH₂, 2H); 5.760 (s, CH, 1H); 5.800 (s, CH,1H); 7.100-8.450 (m, aromatic, 12H).

Compound 3: Deprotection of the amino group in 3d was carried out usingmethylamine in methanol by stirring a solution of the parent dye inmethanol with 1 M methylamine in methanol. The product was isolated byevaporation of the solvent. The deprotected amine derivative 3e of thedye was reacted with iodoacetic anhydride (1:1) in methylene chloride,and the final product was isolated by filtration. The final dye 3 waspurified by repeated precipitation using methylene chloride and hexane.

¹H NMR (DMSO-d₆, TMS) δ ppm: 1.420 (t, CH₃, 3H); 3.900 (s, CH₂, 2H);4.300 (q, CH₂, 2H); 4.450 (t, CH₂, 2H); 4.480 (t, CH₂, 2H); 5.820 (s,CH, 1H); 5.980 (s, CH, 1H); 7.200-8.200 (m, aromatic, 8H).

Example 4

In this example, thiol-reactive Nile Red nulceus 4 was preparedaccording to Scheme III.

Intermediate 4b: N-phenyl-N-methyl-ethanolamine 4a (50 mmol) wassuspended in conc. HCl (28 mL) and was cooled to 5° C. To this solutionwas added dropwise a sodium nitrite solution (6.67 g in 10 mL water)over a period of 40 min. After the addition, the reaction was keptstirring for 2 h more. The product was then filtered, washed with 0.5 MHCl, and dried in vacuo to give the nitroso compound 4b.

¹H NMR (D₂O) δ ppm: 3.59 (s, CH₃, 3H); 3.90 (t, CH₂, 2H); 4.05 (t, CH₂,2H); 7.22-7.30 (m, aromatic, 2H); 7.50 (d, aromatic, 1H); 7.77 (d,aromatic, 1H). 13C NMR (D₂O) δ ppm: 42.44, 57.92, 58.72, 120.29, 122.52,125.93, 140.56, 149.93, 163.21.

Mol. Wt calculated for C₉H₁₂N₂O₂ is 180(M+), found 181 (M+1) (FAB)

Intermediate 4d: 1,3-dihydroxynaphthalene 4c (5 mmol) was suspended inethanol (25 mL) and was brought to reflux while stirring. To therefluxing solution was added intermediate 4b (5 mmol) in fractions overa period of 45 min. After the addition, the reaction mixture wasmaintained at reflux for 4 h more and was then cooled. The solvent wasevaporated, and the product dye was purified by flash columnchromatography over silica gel using methanol and chloroform (1:9) aseluent.

¹H NMR (CDCl₃) δ ppm: 2.98 (s, 3H), 3.48 (t, 2H), 3.83 (t, 2H), 6.20 (s,1H), 6.77 (d, 1H), 7.10 (d, 1H), 7.45 (s, 1H), 7.60-7.75 (m, 3H), 7.72(m, 1H), 8.08 (m, 1H). ¹³C NMR (CDCl₃) δ ppm: 39.2, 55.6, 60.3, 102.2,110.2, 113.6, 124.7, 126.3, 126.6, 126.8, 130.6, 132.3, 133.7, 135.1,145.8, 148.2, 152.4, 182.5, 183.8.

Mol. Wt calculated for C₁₉H₂₆N₂O₃ is 320(M+), found 321 (M+1) (FAB)

Compound 4: The intermediate dye 4d was dissolved in anhydrousacetonitrile (10 mL), and p-dimethylaminopyridine (3 mg) was added,followed by iodoacetic anhydride (250 mg). The reaction was stirred for2 h. The product 4 was separated by evaporation of the solvent and thenpurified by repeated precipitation from methylene chloride and hexane.

¹H NMR (CDCl₃) δ ppm: 3.14 (s, 3H), 3.66 (s, 2H), 3.74 (t, 2H), 4.38 (t,2H), 6.40 (s, 1H, ArH), 6.54 (d, 1H, ArH), 6.72 (d, 1H, ArH), 7.64-7.75(m, 3H, ArH), 8.29 (d, 1H, ArH), 8.65 (d, 1H, ArH). ¹³C NMR (CDCl₃) δppm: −6.1, 39.6, 50.9, 63.0, 97.5, 106.4, 110.1, 124.1, 125.6, 126.0,130.6, 131.2, 131.7, 131.98, 132.0, 141.5, 146.5, 151.8, 152.2, 169.0,184.1.

Mol. Wt calculated for C₂₁H17IN₂O₄ is 488, found 489 (MH⁺) (FAB-MS).

Example 5

This example produced Nile Red nucleus 5 according to the process ofScheme IV.

Intermediate 5b: 9-Diethylamino-2-hydroxy-5H-benz[a]phenoxazin-5-one 5a(50 mg, 0.15 mmol), N-bromoethylphthalimide (50 mg, 0.20 mmol) andpotassium carbonate (60 mg, 0.09 mmol) were combined in DMF (15 mL)under argon with stirring. The reaction proceeded at reflux for 4.5 h.Additional bromoethylphthalimide (25 mg) was added at 4.5 h and at 6.5 h(15 mg). The temperature was lowered to 115° C., and the reactionproceeded overnight. DMF was removed in vacuo, and the residue was driedin vacuo. Column chromatography (2% MeOH/CH₂Cl₂) afforded 38 mg of theproduct 2-(2-phthalimidylethoxy)-Nile Red 5b.

¹H NMR (CDCl₃) δ ppm: 1.22 (t, 6H), 3.40 (q, 4H), 4.19 (t, 2H), 4.40 (t,2H), 6.23 (s, 1H), 6.36 (d, 1H), 6.59 (dd, 1H), 7.09 (d, 1H), 7.52 (d,1H), 7.70 (m, 2H), 7.84 (m, 2H), 7.96 (d, 1H), 8.13 (d, 1H). ¹³C NMR(CDCl₃) δ ppm: 12.8, 37.5, 45.2, 65.3, 96.3, 105.2, 106.5, 109.8, 118.6,123.5, 124.9, 126.0, 127.9, 131.3, 132.1, 134.2, 139.6, 146.8, 150.7,152.2, 161.0, 162.8, 168.4, 183.3. Mol. Wt calculated for C₃₀H₂₅N₃O₅ is507, found 508 (MH⁺) (FAB-MS).

Intermediate 5c: Intermediate 5b (30 mg, 0.06 mmol) was dissolved inanhydrous MeOH (8 mL) and was placed under argon. Next, methylamine (2Min MeOH, 4 mL, 8 mmol) was added. The reaction was carried out for 5 minat RT and 2.5 h at reflux. Flash chromatography was performed in 10%MeOH/CH₂Cl₂ to remove fast moving impurities and then at 30% MeOH/CH₂Cl₂to elute the product 2-(2-aminoethoxy)-Nile Red 5c. Solvent was removedon a rotary evaporator, and the residue was dried in vacuo. Yield: 11mg.

¹H NMR (CDCl₃) δ ppm: 1.25 (t, 6H), 3.17 (t, 2H), 3.45 (q, 4H), 4.20 (t,2H), 6.27 (s, 1H), 6.43 (d, 1H), 6.63 (dd, 1H), 7.15 (m, 1H), 7.56 (d,1H), 8.02 (m, 1H), 8.18 (d, 1H). ¹³C NMR (CDCl₃) δ ppm: 12.8, 41.6,45.3, 63.9, 96.4, 105.4, 106.6, 109.9, 118.4, 124.9, 125.9, 127.4,128.0, 131.3, 134.3, 147.1, 151.0, 152.3, 161.7, 183.5.

Mol. Wt calculated for C₂₂H₂₃N₃O₃ is 377, found 378 (MH⁺) (FAB-MS).

Compound 5: Intermediate 5c (11 mg, 0.03 mmol) was dissolved in CH₂Cl₂(5 mL). lodoacetic anhydride (21 mg, 0.06 mmol) was then added andallowed to react for 40 min. An additional 25 mL of CH₂Cl₂ was added,and the reaction mixture was transferred to a separatory funnel. Theorganic phase was washed twice with 10% Na₂CO₃ (10 mL each), dried overanhydrous MgSO₄, and filtered. After the solvent was removed on a rotaryevaporator, the final product 2-iodoacetylamidoethoxy-Nile Red nucleus 5was dried under vacuum and precipitated from CH₂Cl₂/hexane. Yield: 3.4mg.

¹H NMR (CDCl₃) δ ppm: 1.17 (t, 6H), 3.35 (q, 4H), 3.69 (t, 2H), 3.78 (s,2H), 4.14 (t, 2H), 6.12 (s, 1H), 6.27 (s, 1H), 6.48 (dd, 1H), 6.91 (d,1H), 7.34 (d, 1H), 7.74 (s, 1H), 7.95 (s, 1H). ¹³C NMR (CDCl₃) δ ppm:12.9, 29.9, 40.1, 45.3, 66.9, 96.3, 105.2, 106.7, 109.8, 118.0, 124.9,125.9, 127.8, 131.3, 134.1, 139.4, 147.0, 151.0, 152.2, 161.1, 168.1,183.3.

Mol. Wt calculated for C₂₄H₂₄IN₃O₄ is 545, found 546 (M−H⁺) (CI-MS).

Example 6

This example produces iodoacetamido benzodioxazole nucleus 6 accordingto Scheme V.

Intermediate 6b: Aminobenzodioxazole 6a (10 mmol) was reacted with ethylbromide (50 mmol) in the presence of anhydrous potassium carbonate. Theproduct was purified by column chromatography over silica gel usingchloroform and methanol to afford the intermediate 6b in 65% yield.

Mol. Wt calculated for C₁₀H₁₃N₃O is 191 (M+), found 191 (M+1) (FAB)

Intermediate 6c: POCl₃ (1 mL) was added to anhydrous DMF (4 mL) kept at˜5° C. in a round-bottomed flask with stirring. To this mixture wasadded intermediate 6b (0.4 g), and stirring continued for 1 h. Thereaction was quenched by adding the reaction mixture to ice water (100mL), followed by neutralization with 1N KOH (pH adjusted to ˜9.0). Theproduct was extracted with methylene chloride, and the organic phase wasdried over sodium sulfate. The product was purified by columnchromatography over silica gel using chloroform to afford 85% of theintermediate 6c.

¹H NMR (CDCl₃) δ ppm: 1.36 (t, CH₃, 6H); 3.91 (q, CH₂, 4H); 6.19 (d, CH,1H); 7.82 (d, CH, 1H); 10.01 (s, CH, 1H).

Mol. Wt calculated for C₁₁H₁₃N₃O₂ is 219(M+), found 220 (M+1) (FAB)

Intermediate 6e: Intermediate 6c (350 mg) was reacted with intermediate6d (644 mg, prepared in the same manner as intermediate 2b) in anhydrousmethanol under reflux for 6 h in the presence of piperidine (50 mg) toform the parent dye 6e. The crystals that separated upon cooling werecollected by filtration and then were purified by flash columnchromatography over silica gel using a mixture of methanol (5%) andchloroform.

¹H NMR (CDCl₃, TMS) δ ppm: 1.42 (t, CH₃, 6H); 3.20 (t, 2H); 4.0 (q, CH₂,4H); 4.40 (t, CH₂, 2H); 6.50 (s, CH, 1H); 6.51 (s, CH, 1H); 7.5-8.7 (m,aromatic, 10H).

Mol. Wt calculated for C₂₉H₂₆N₅O₃S is 524(M+), found 524 (FAB)

The obtained dye 6e showed a polar sensitivity as in Table 1 below:

TABLE 1 Solvent Relative Fluorescence Methylene Chloride 100Acetonitrile 62 Ethanol 66 Methanol 50 Water 1

Such solvent polarity sensitivity of a dye is indicative of itsenvironmental-sensitivity when attached to protein.

The parent intermediate benzodioxazole nucleus 6e can be deprotectedusing Na₂S and water to produce dye 6f and subsequently reacted withiodoacetic anhydride to form the target dye 6.

Example 7

This example produces iodoacetamido benzodioxazole nucleus 7 accordingto Scheme VI. Intermediate 7c was produced in the same manner as 6c fromExample 6.

Intermediate 7d: 4-picoline (1 g) was reacted with 2-bromoethylphthalimide (2.5 g) by heating at 125° C. for 12 h. The colorless solidformed was purified by repeated washing with chloroform to yield 3 g(86%) of the compound 7d.

¹H NMR (CD₃OD) δ ppm: 2.67 (s, CH₃, 3H); 4.30 (t, CH₂, 2H); 4.88 (t,CH₂, 2H); 7.81 (m, 4H); 7.93 (d, 2H); 8.93 (m 2H). ¹³C NMR (CD₃OD) δppm: 22.12, 39.63, 60.69, 124.49, 130.00, 132.90, 135.76, 145.45,162.06, 169.20.

Intermediate 7e: Intermediate 7c (220 mg) was reacted with intermediate7d (347 mg) in anhydrous methanol under reflux for 6 h in the presenceof piperidine (50 mg) to form the parent dye 7e. The crude product wassubjected to column chromatography over silica gel using methanol andchloroform (1:9) to obtain 7e.

Compound 7: Deprotection of the phthalimide in 7e provides intermediate7f, which is then reacted with iodacetic anhydride to produce the finalproduct, compound 7.

Example 7-1

This example can be used to produce the iodoacetyl benzodioxazolenucleus 7′ according to reaction Scheme VIa.

Compound 7a′ is reacted with an equivalent amount of methyl iodide inpresence of potassium carbonate and a phase transfer catalyst to formintermediate 7b′. A subsequent reaction of intermedicate 7b′ with2-bromoethanol produces intermediate 7c′. Vilsmaeir reaction onintermediate 7c′ produces intermediate 7d′, and a reaction of 7d′ with7e′ produces intermediate 7f. A reaction of 7f with iodoacetic anhydridewill produce the final compound 7′.

Example 8

This example produces the iodoacetyl benzodioxazole nucleus 8 accordingto Scheme VII.

Intermediate 8b: Aminobenzodioxazole 8a (10 mmol) was reacted with ethylbromide (50 mmol) in the presence of anhydrous potassium carbonate. Theproduct was purified by column chromatography over silica gel usingchloroform and methanol to afford the intermediate 8b in 65% yield.

Mol. Wt calculated for C₁₀H₁₃N₃O is 191 (M+), found 191 (M+1) (FAB)

Intermediate 8c: POCl₃ (1 mL) was added to anhydrous DMF (4 mL) kept at˜5° C. in a round-bottomed flask with stirring. To this mixture wasadded intermediate 8b (0.4 g), and stirring continued for 1 h. Thereaction was quenched by adding the reaction mixture to ice water (100mL), and was followed by neutralization with 1N KOH (pH adjusted to˜9.0). The product was extracted with methylene chloride, and theorganic phase was dried over sodium sulfate. The product was purified bycolumn chromatography over silica gel using chloroform to afford 85% ofthe intermediate 8c.

¹H NMR (CDCl₃) δ ppm: 1.36 (t, CH₃, 6H); 3.91 (q, CH₂, 4H); 6.19 (d, CH,1H); 7.82 (d, CH, 1H); 10.01 (s, CH, 1H).

Mol. Wt calculated for C₁₁H₁₃N₃O₂ is 219(M+), found 220 (M+1) (FAB-MS).

Intermediate 8d: A mixture of 2-methylbenzothiazole (2.24 g, 15 mmol)and 2-bromoethanol (2.90 g, 23 mmol) was taken in 25 mL flask. Thereaction mixture was heated at 120° C. for 24 h. After 24 h the reactionmixture was cooled to room temperature and chloroform (20 mL) was addedand stirred for 4 h at room temperature. The solid product was filtered,washed with chloroform and dried to give the desired product 8d as lightbrown solid. ¹H NMR (CD₃OD) δ ppm: 3.26 (s, 3H), 4.06 (t, 2H), 4.94 (t,2 H), 7.82 (t, 1H), 7.90 (t, 1H), 8.25-8.32 (m, 2H). ¹³C NMR (CD₃OD) δppm: 16.8, 52.3, 59.1, 117.0, 124.2, 128.5, 129.4, 129.7, 141.6, 178.1.

Intermediate 8e: Intermediate 8c (55 mg) was reacted with intermediate8d (70 mg) in anhydrous methanol under reflux for 5 h in the presence ofpiperidine (100 mg) to form the parent dye 8e. The crystals thatseparated upon cooling were collected by filtration and confirmed by NMRand mass spectroscopy. ¹H NMR (CD₃OD) δ ppm: 1.38 (t, 6H), 3.13 (t, 2H),4.03 (q, 4H), 4.13 (t, 2H), 6.51 (d, 1H), 6.53 (d, 1H), 7.67 (m, 1H),7.78 (m, 1H), 7.89 (m, 1H), 8.03 (m, 1H), 8.14 (m, 1H), 8.20 (m, 1H).¹³C NMR (CD₃OD) δ ppm: 11.62, 44.54, 51.08, 59.18, 104.72, 108.01,109.18, 115.96, 123.33, 127.47, 127.70, 129.09, 142.10, 143.40, 144.25,144.97, 145.35, 149.04, 172.69.

Mol. Wt calculated for C₂₉H₂₆N₅O₃S is 395(M+), found 395 (FAB MS).

Compound 8: The parent dye 8e (20 mg) was reacted with iodoaceticanhydride (20 mg) by stirring at room temperature (3 h) in anhydrousmethylene chloride (5 mL) in presence of pyridine (50 mg). The obtainedproduct dye 8 was purified by precipitation from hexane.

Mol. Wt calculated for C₂₃H₂₄IN₄O₃S⁺ is 563 (M+), found 563 (FAB)

Example 8-1

This example produces the iodoacetyl benzodioxazole nucleus 8′ accordingto reaction scheme VIIa.

Compound 8a′ is reacted with an equivalent amount of methyl iodide inthe presence of anhydrous potassium carbonate and a phase transfercatalyst to form the mono-methylamino derivative 8b′. Compound 8b′ isthen reacted with 2-bromoethanol in presence of potassium carbonate andphase transfer catalyst to form intermediate 8c′. Vilsmaeir reaction on8c′ produces intermediate 8d′, which is reacted with 8e′ to formintermediate 8f. A subsequent reaction of intermediate 8f withiodoacetic anhydride produces the final compound 8′.

Example 9

This example produces the aza coumarin nucleus 9 according to schemeVIII.

Intermediate 9b. A solution of N,N′-dimethylamino phenol 9a (3.42 g, 25mmol) in 10 mL of concentrated HCl was placed in a 100 mL flask and themixture was cooled to 5° C. The content in the flask was stirredvigorously and a solution of NaNO₂ (1.80 g, 26 mmol) in water (5 mL) wasadded directly into the reaction mixture over a period of 30 min. Thereaction temperature was maintained at 5° C. throughout the period ofaddition. After the addition was over, the reaction mixture was stirredfor 1 h at 5° C. and filtered. The solids were washed with 10 mL of 5MHCl followed by ethanol (25 mL) and dried in air to give a yellow solid9b (3.00 g, 72%).

Mol. Wt calculated for C₈H₁₀N₂O₂ is 166.18, found 167 (MH⁺) (FAB-MS).

Intermediate 9e. A slurry of 10% Pd/C (25 mg) in 5 mL of methanol wasstirred for 15 min. under an atmosphere of argon. A solution of NaBH₄(190 mg, 5.0 mmol) in methanol (5 mL) was added to this slurry. Asolution of the nitroso compound 9b (500 mg, 2.5 mmol) dissolved inmethanol (13 mL) and containing triethylamine (2 mL) was added dropwiseto the Pd/C slurry over a period of 5 min. The red nitroso compoundturned into light yellow during the reduction reaction. After 15 min, anadditional amount of NaBH₄ (190 mg, 5 mmol) in methanol (4.0 mL) wasadded to ensure complete reduction to form intermediate 9c. Stirring wascontinued for another 30 min, ethylpyruvate 9d (3.0 mL, 27 mmol) wasadded to the reaction mixture, and then the contents were heated toreflux. After 3 h of refluxing, the reaction mixture was cooled to roomtemperature and filtered through celite to remove the unreacted Pd/C.The filtrate was evaporated to obtain a residue, which waschromatographed over silica gel and eluted with a mixture of hexane andethyl acetate (9:1 v/v), to give a yellow solid 9e (400 mg, 78%). ¹H NMR(CDCl₃) δ ppm: 2.48 (s, 3H), 3.06 (s, 6H), 6.40 (s, 1H), 6.65 (m, 1H),7.50 (m, 1H). ¹³C NMR (CDCl₃) δ ppm: 21.0, 40.5, 97.5, 109.8, 122.9,129.2, 147.6, 148.9, 152.0, 154.7.

Mol. Wt calculated for C₁₁H₁₂N₂O₂ is 204, found 205 (MH⁺) (FAB-MS).

Intermediate 9g. POCl₃ (1.13 g, 7.4 mmol) was added to anhydrous DMF (15mL) kept at 5° C. The mixture was stirred under an atmosphere of argonfor 30 min. at 5° C. and then 2-(methylphenylamino)-ethanol (1.12 g, 7.4mmol) 9f was added and the resulting solution was stirred at roomtemperature for 3 h. The reaction mixture was hydrolyzed by slowaddition of ice-cold water and neutralized by the addition of NaOH (2 M)and the pH was adjusted to 7.0. The product was extracted with methyltert-butyl ether (3×50 mL), dried over anhydrous Na₂SO₄ and evaporatedto give a yellow liquid. The product was purified by columnchromatography as follows. The crude product was chromatographed oversilica gel and eluted with a mixture of hexane and ethyl acetate, (9:1v/v). Evaporation of the pure fractions yielded compound 9g (0.80 g,60%). ¹H NMR (CDCl₃) δ ppm: 3.00 (s, 3H), 3.64 (t, 2H), 4.36 (t, 2H),6.76 (m, 2H), 7.28 (m, 2H), 8.07 (s, 1H). ¹³C NMR (CDCl₃) δ ppm: 38.9,51.2, 61.3, 112.4, 117.1, 129.5, 149.0, 161.2.

Mol. Wt calculated for C₁₀H₁₃NO₂ is 179, found 179 (M+) (FAB-MS).

Intermediate 9h. A solution of compound 9e (100 mg, 0.49 mmol) andcompound 9g (100 mg, 0.56 mmol) in anhydrous methanol (4 mL) was stirredunder an atmosphere of argon. To this solution, sodium methoxide (26 mg,0.50 mmol) was added and the reaction mixture was heated to reflux.After 4 h, the reaction mixture was cooled to room temperature andevaporated to afford a dark brown residue. The obtained residue wasdissolved in minimum amount of chloroform and chromatographed oversilica gel. Elution with a mixture of methanol and chloroform (5:95,v/v) yielded the product 9h as a solid (40 mg, 23%). ¹H NMR (CDCl₃) δppm: 2.96 (s, 6H), 3.10 (s, 3H), 3.47 (t, 2H), 3.82 (t, 2H), 6.36 (s,1H), 6.46 (s, 1H), 6.53 (d, 1H), 6.75 (m, 1H), 6.80 (d, 2H), 7.24 (m,2H), 7.56 (d, 1H). ¹³C (CDCl₃) δ ppm: 38.9, 40.6, 55.7, 60.3, 96.9,102.2, 103.8, 111.0, 113.3, 117.4, 129.4, 129.8, 143.1, 144.5, 145.3,149.7, 150.3, 151.8, 178.88.

Compound 9 (Iodoacetylaza-coumarin, IAZCO). To a solution of compound 9h(20 mg, 0.060 mmol) in anhydrous chloroform (2 mL), pyridine (50 μL) wasadded and stirred under an atmosphere of argon for 5 min. To thissolution was added iodoaceticanhydride (30 mg, 0.09 mmol) and thestirring was continued for 2 h. Chloroform was evaporated off and theresidue was chromatographed over silica gel and eluted with a mixture ofmethanol and chloroform (5:95, v/v) to give the product 9 as a darksolid. (25 mg, 85%).

Example 9-1

This example produces the azacoumarin nucleus 9′ according to thereaction scheme VIIIa.

3-hydroxy-N-methylaniline 9a′ is reacted with sodium nitrite in presenceof HCl to produce the compound intermediate 9b′. Reduction of thenitroso group of 9b′ is carried out with Pd/C and sodium borohydride toproduce the intermediate 9c′, followed by reaction with 9d′ to produceintermediate 9e′. A reaction of 2-bromoethanol with intermediate 9e′produces intermediate 9f′. Intermediate 9f′ is reacted with 9g′ toproduce intermediate 9h′. The final product 9′ is obtained by reacting9h′ with iodoacetic anhydride in presence of pyridine.

Example 10

In this Example, reaction Scheme IX was used to produce the Compound 10.

Intermediate 10c. 4-(diethylamino)salicylaldehyde 10a (2.00 g, 10.0mmol) and diethyl glutaconate 10b (2.00 g, 11.0 mmol) were refluxed inabsolute ethanol (25 mL) in presence of piperidine (50 mg). After 6 hrefluxing, the reaction mixture was cooled to room temperature and theyellow crystals obtained was separated, washed with cold ethanol (10 mL)and dried under vacuo to obtain the desired product 10c (2.60 g, 82%).¹H NMR (CDCl₃) δ ppm: 1.22 (t, 6H), 1.29 (t, 3H), 3.42 (q, 4H), 4.23 (q,2H), 6.48 (d, 1H), 6.60 (d, 1H), 6.94 (d, 1H), 7.30 (d, 1H), 7.53 (d,1H), 7.70 (s, 1H). ¹³C NMR (CDCl₃) δ ppm: 12.7, 14.6, 45.2, 60.6, 97.2,108.8, 109.6, 114.8, 119.7, 130.0, 139.6, 144.6, 151.9, 156.7, 160.5,168.0.

Mol. Wt calculated for C₁₈H₂₁NO₄ is 315, found 315 (M⁺) (FAB-MS).

Intermediate 10d. Water (3 mL) was added to a solution of compound 10c(1.00 g, 0.315 mmol) in THF (12 mL). To this mixture, 20 mg of OsO₄(2.5% in t-butanol) was added and the obtained reaction mixture wasstirred at room temperature for an hour. After this period, portions ofpowdered NaIO₄ (1.50 g, 7.0 mmol) were added over a period of 30 min andstirring continued for an additional 48 h. After stirring, the solventwas removed and the obtained solid was dissolved in CH₂Cl₂ (200 mL) andwashed with water (75 mL). The organic layer was washed with brine,dried over sodium sulfate and evaporated to obtain a yellow solid. Thecrude product containing small amounts of starting material was purifiedby silica gel column chromatography (CH₂Cl₂/EtOAc, 4:1) to yield thepure product 10d as a yellow solid (320 mg, 41%). ¹H NMR (CDCl₃) δ ppm:1.25 (t, 6H), 3.48 (q, 4H), 6.48 (d, 1H), 6.64 (m, 1H), 7.40 (d, 1H),8.25, (s, 1H), 10.12 (s, 1H). ¹³C NMR (CDCl₃) δ ppm: 12.67, 45.49,97.35, 108.43, 110.39, 114.50, 132.4, 145.6, 153.7, 159.2, 162.1, 188.2.

Mol. Wt calculated for C₁₄H₁₅NO₃ is 245, found 246 (MH⁺) (FAB-MS).

Intermediate 10f. A mixture of 2-methylbenzothiazole 10e (2.24 g, 15mmol) and 2-bromoethanol (2.90 g, 23 mmol) was taken in 25 mL flask. Thereaction mixture was heated at 120° C. for 24 h. After 24 h the reactionmixture was cooled to room temperature, and chloroform (20 mL) was addedand stirred for 4 h at room temperature. The solid product was filtered,washed with chloroform and dried to give the desired product 10f aslight brown solid. ¹H NMR (CD₃OD) δ ppm: 3.26 (s, 3H), 4.06 (t, 2H),4.94 (t, 2H), 7.82 (t, 1H), 7.90 (t, 1H), 8.25-8.32 (m, 2H). ¹³C NMR(CD₃OD) δ ppm: 16.8, 52.3, 59.1, 117.0, 124.2, 128.5, 129.4, 129.7,141.6, 178.1.

Intermediate 10g. A solution of coumarin aldehyde 10d (60 mg, 0.22 mmol)and compound 10f (50 mg, 0.20 mmol) in anhydrous methanol (3 mL) wastaken in a 10 mL flask. To this solution piperidine (30 mg) was addedand heated to reflux. The light brown reaction mixture slowly changed toviolet in about 30 min. The contents were refluxed overnight, cooled toroom temperature and purified by column chromatograpy over silica gelusing chloroform containing 3% methanol as the solvent. Evaporation ofthe pure fractions yielded 45 mg of the desired dye, 10g.

Compound 10 (ICOBzT). To a solution of compound 10g (30 mg, 0.060 mmol)in anhydrous chloroform (3 mL), was added pyridine (30 mg) and stirredunder an atmosphere of argon. lodoacetic anhydride (30 mg, 0.080 mmol)was added to this stirred reaction mixture and continued the stirringfor 3 h. The solvent was then evaporated and the obtained residue waschromatographed over silica gel. Elution with chloroform containing 5%methanol yielded the pure compound 10 as a purple solid.Glucose/galactose mutant A213C conjugated to 10 (ICOBzT) as describedherein, showed a wavelength shift of 10 nm (red shift) in presence of100 mM of glucose. (shifted from 622 to 632 nm).

Example 10-1

This example produces the coumarin nucleus 10′ according to reactionscheme IXa.

3-hydroxy-N-ethylaniline (10a′) is reacted with 2-bromoethanol toproduce intermediate 10b′. Vilsmaeir reaction is carried out on 10b′ toobtain intermediate 10c′. Further reaction of 10c′ with 10d′ producesintermediate 10e′. Reaction with OsO₄ and sodium periodate producesintermediate 10f′. Subsequently, 10f′ is reacted with 10g′ to produceintermediate 10h′. The desired product 10′ is obtained by the reactionof 10h′ with iodoacetic anhydride.

Example 11

GGBP Conjugation. A solution of H152C GGBP (4 nmol) in 200 uL PBS bufferwas prepared, and this was incubated with DTT (8 nmol) for 30 minutes atroom temperature. A solution of the squaraine iodoester nucleus 1 (1 mg,partially dissolved in 120 uL DMSO) was added, and the mixture waswrapped in foil and left for 4 h at room temperature. The labeledprotein was obtained as the second fraction from a NAP-5 size exclusioncolumn, eluting with PBS buffer. The protein was assayed for itsfluorescence response to glucose in several wells of a 96 well microwellplate with glucose added in PBS giving final glucose concentrationsbetween 0 and 1 mM. The fluorescence response of the labeled protein toglucose was determined by adding glucose to a solution of the labeledprotein in PBS buffer. Typically the fluorescence measurements employedeither a Varian Cary Eclipse fluorimeter (Varian, Inc., Palo Alto,Calif.) or a PTI spectrofluorimeter (Photon Technology International,Inc., Lawrenceville, N.J.). The plate fluorescence was read using aVarian Cary Eclipse fluorometer equipped with a microwell plate adapterusing excitation at 625 nm and emission at 660 nm. Thus, the squaraineiodoester nucleus 1-binding protein conjugate fluorescence propertycorresponded to analyte concentration and therefore functioned as abiosensor. This indicated an approximate Kd of 6 uM between the labeledprotein and glucose as shown in FIG. 3.

Further labeling of individual GGBP variants was performed with thesquaraine iodoester 1. Binding constants (Table 2) were determined bypreparing samples with approximately 0.1 μM labeled protein in buffer(PBS) in a 96 well microplate and adding solutions of varyingconcentrations of glucose (giving final concentrations between 0 and 1mM or between 0 and 10 mM). The K_(d) was determined from the followingrelationships as adapted from M. L. Pisarchick and N. L. Thompson“Binding of a monoclonal antibody and its Fab fragment to supportedphospholipid monolayers measured by total internal reflectionfluorescence microscopy” Biophys. J. 1990, 58, 1235-1249: where F isfluorescence intensity, F_(inf) is fluorescence at infinity, F₀ isfluorescence at zero glucose, and x is the free concentration of glucose([Glc]_(free)) as determined by the relationship:

$\lbrack{GLc}\rbrack_{free} = \frac{\begin{matrix}{\lbrack{GLC}\rbrack_{tot} - \lbrack{Prot}\rbrack_{tot} - {Kd} +} \\\sqrt{\left( {\lbrack{Glc}\rbrack_{tot} - \lbrack{Prot}\rbrack_{tot} - {Kd}} \right)^{2} + {4*\lbrack{Glc}\rbrack_{tot}*{Kd}}}\end{matrix}}{2}$where [Glc]_(tot) and [Pro]_(tot) are the total concentrations ofglucose and protein, respectively. Note that when [GLc]_(tot)>>Kd and[GLc]_(tot)>>[Pro]_(tot), the above two equations may be simplified tothe following form:F=F ₀+[(F _(const) *x)/(1+x/Kd)]where F_(const)=(F_(inf)−F₀)/K_(d).

TABLE 2 Fluorescence Intensity Mutant GGBP Change (%) Kd (mM) forGlucose H152C GGBP +100% 0.006 E149C/A213R GGBP +100% 0.068 A213C/L238CGGBP +130% 0.75

Example 12

Several conjugates of dye 2 with GGBP (glucose/galactose bindingprotein) were prepared that had cysteine substitutions in the protein asidentified in Table 3. In general, conjugates of compound 2 with GGBPwere substantially more stable in solution than conjugates of compound 1with GGBP. An aliquot of the protein in PBS buffer was treated with DTT(dithiothreitol) for 10-30 minutes followed by addition of theiodoacetyl squaraine dye in DMSO. After approximately 3-4 hours thereaction was stopped, and dye-labeled protein was obtained bysize-exclusion chromatography (NAP-5 column). The fluorescence responsewas determined with excitation at 600 nm and emission scanned between625 nm and 700 nm. Emission maxima were observed near 660 nm.

The absorption spectrum for the conjugate of compound 2 with V19C GGBPis shown in the graph of FIG. 4. This absorption spectrum is typical forthe conjugates described herein.

All the conjugates were treated with at least 10 mM of glucose, and thefluorescence changes were monitored. FIG. 5 illustrates the change influorescence observed with change in glucose concentration for compound2 conjugated to V19C GGBP. For the derivative A213C, a 2-3 nmfluorescence shift to the blue region was observed upon glucose binding.Thus, the squaraine iodoester nucleus 2-binding protein conjugatefluorescence property corresponded to analyte concentration andtherefore functioned as a biosensor.

TABLE 3 Fluorescence Intensity GGBP Mutant Change (%) E149C/A213R/L238S+5% K11C −5% K113C −15% V19C +25% W183C +17% D236C +15% M182C −14% T110C−15%

Example 13

The same protocol was used to label E149C GGBP, H152C GGBP and S337C MBPwith the Nile Red compounds 4 and 5. Fluorescence excitation was at 550nm, and the emission maximum was 650 nm. The change in fluorescenceintensity is shown in Table 4. Thus, the Nile Red nucleus 4- and5-binding protein conjugate fluorescence property corresponded toanalyte concentration and therefore functioned as a biosensor.

TABLE 4 Fluorescence Nile Red-Binding Protein Intensity Conjugate change(%) E149C GGBP with compound 5 +9% H152C GGBP with compound 5 +6% S337CMBP with compound 4 +200%

Example 14

Glucose/Maltose sensing using 9 (IAZCO). The aza-coumarin nucleus IAZCOwas conjugated to glucose binding protein (GBP) and maltose bindingprotein (MBP) as described in the previous examples. Derivatives of theprotein GGBP (glucose/galactose binding protein) and MBP (maltosebinding protein) with cysteine residue substitutions were prepared.Typically, an aliquot of the protein in PBS buffer was treated with DTT(dithiothreitol) for 10-30 minutes followed by addition of the dye 9 inDMSO. After approximately 3-4 hours, the reaction was stopped anddye-labeled protein was obtained by size-exclusion chromatography (NAP-5column). The fluorescence response of the labeled protein toglucose/maltose was determined by adding glucose/maltose with excitationat 600 nm and emission scanned between 620 nm and 700 nm. Thus,glucose/galactose binding protein mutant E149C conjugated to 9 (IAZCO)showed a wavelength shift of 9 nm (blue shift) in presence of 100 mMglucose (shifted from 653 nm to 644nm) and thus functioned as abiosensor.

The fluorescence changes observed for 9 with different proteins andglucose/maltose are given in Table 5. These results were obtained bymeasuring the fluorescence of the dye-protein conjugate (<1.0 μM) in PBS(at pH 7.4). Saturation amounts (100 mM) of glucose/maltose were added,and the ratio of the fluorescence was obtained. Thus, the aza-coumarinnucleus 9-binding protein conjugate fluorescence property correspondedto analyte concentration and therefore functioned as a biosensor.

TABLE 5 Change in GGBP/MBP Fluorescence Conc. of Mutant Intensityglucose/Maltose Kd (mM) E149C GGBP +60% 100 mM -na A213C GGBP +130% 100mM 0.013 H152C GGBP +75% 100 mM -na V19C GGBP −50% 100 mM -na E149C,A213C, L238S +100% 100 mM 30    GGBP S337C MBP +1100% 100 mM 0.060

Example 15

Reading Through Skin Experiment

In one embodiment, in vitro through skin glucose/maltose sensingexperiments were performed using the herein described NIR dyes.

Three protein-NIR conjugates (shown below) were chosen as the testsubstrates.

-   -   (a) A213C GGBP conjugated to iodoacetyl aza-coumarin nucleus        (IAZCO) of Compound 9    -   (b) S337C MBP conjugated to iodoacetyl aza-coumarin nucleus        (IAZCO) of Compound 9    -   (c) S337C MBP conjugated to iodoacetyl Nile red nucleus (INR) of        Compound 4

These conjugates were infused into crosslinked polyethylene glycol (PEG)disks and the disks were used for read-through-skin studies. The PBSsolutions of these conjugates in micro-well plates were also studiedthrough skin. A blank PEG disk and PBS were used as the control. Theactivity of the proteins in PBS and PEG disks were tested prior to invitro experiment.

(i) Response to Glucose

Referring to FIG. 6, the PEG disks 10 were placed over a piece of rabbitskin 12 and was excited by a laser 14 and read from the other side ofthe skin by a detector 16.The Rabbit skin was about 3-4 mm thick and wasnot transparent to human eye. Excitation was carried out using 590 nmLED light and the fluorescence was monitored at 650 nm. Filters 18 wereused to avoid interference from the excitation light and scattering.

The fluorescence was stable for both PEG disks and solutions, and uponaddition of glucose the fluorescence intensity rose and kept increasingfor 2-4 minutes. This time lag may represent a slow diffusion ofglucose/maltose in to the hydrogel as shown in FIG. 7. For the solutioncontrol, the response was fast and no lag was noticed. Control studiesusing blank PEG disk and PBS solutions showed no change in fluorescenceupon addition of glucose. Thus, the aza-coumarin nucleus 9-bindingprotein conjugate, and Nile Red nucleus 4-binding protein conjugatefluorescence property corresponded to the presence of analyte andtherefore functioned as a through-skin biosensor.

(ii): Response to Maltose using MBP

The above experiments were repeated using the INR-MBP PEG disks andsolutions. As in the case of A213C GGBP conjugated to 9, thefluorescence was stable for both PEG disks and solutions. Thefluorescence intensity rose and kept increasing for 2-4 minutes uponaddition of maltose.

The following Table 6 summarizes the change in fluorescence intensityobserved for the read-through-skin experiment.

TABLE 6 Change in Change in Fluorescence Fluorescence Solution Change inIntensity of Intensity of Fluorescence PEG disks solution SubstrateIntensity Through-skin Through-skin A213C-IAZCO +120% +96% +96% GGBPS337C-IAZCO +372% +42% +500% MBP S337C-INR MBP +213% +111% +270%

The data provided in these examples demonstrate that the dyes and theconjugates exhibit analyte binding and stability under ambientconditions. While several embodiments have been selected to demonstratethe various embodiments of the invention, it will be understood by thoseskilled in the art that various changes and modifications can be madewithout departing from the scope of the invention as defined in theappended claims.

What is claimed is:
 1. A compound having the formulaA-Y where A is a Nile Red nucleus having a formula selected from thegroup consisting of;

where R⁵, R⁶ and R⁷ are independently methyl, ethyl, or propyl; andwhere Y is

where n is an integer of 1 to 6; A′—CO—R¹, where A′ is —R²O— or—R²N(R³)—, where R² is a C₁ to C₆ alkyl, R³ is H or CH₃, and R¹ isCH₂Cl, CH₂Br, CH₂I; or

where m is an integer of 2 to
 6. 2. The compound of claim 1, wherein R²is a C₂ to C₄ alkyl.
 3. The compound of claim 2, wherein R² is CH₂CH₂.4. The compound of claim 1, wherein R²O is —CH₂CH₂O—.
 5. The compound ofclaim 1, wherein R²N(R³)— is —CH₂CH₂NH—.
 6. The compound of claim 1,wherein said compound is


7. The compound of claim 1, wherein said compound is